Recent Advances and Challenges in the Production of Hydroxylated Natural Products Using Microorganisms

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this review, the authors present the latest research advances in the hydroxylation of natural products such as amino acids, steroids, terpenoids, lipids and phenylpropanoids. The manuscript contains a large number of references that provide a clear insight into the importance of this process as well as the application of the latest biotechnological approaches. However, the structure of the manuscript has numerous shortcomings.

Table 1 is completely uninformative, while the biosynthesis of hydroxylated amino acids is only presented in Table 2 in the rest of the text. Delete Table 1 and present the most important results for each group of natural products separately in tables.

The manuscript also lacks a section giving a brief overview of the bioengineering methods (metabolic engineering, enzyme engineering, genetic engineering, synthetic biology, artificial intelligence technologies) referred to by the authors.

A few illustrations showing the chemical formula of the compound and possible hydroxylation sites would contribute to the quality of the manuscript, especially for complex compounds such as steroids and their derivatives.

Minor revision

Keywords P450 monooxygenase-delete

Line 46 disease-malaria

Line 113-The full name in front of CYP. Add one general sentence about these enzymes.

Line 130-Rewrite the beginning of the sentence

Line 120-Escherichia coli (E. coli)-It is not necessary to put the abbreviations of the names of microorganism species in brackets. Review the entire manuscript. When microorganisms are mentioned, write the full name in the text first and then use the abbreviation.

Line 155-The full name in front of FMOs.

Throughout the text, check that the full names are written before the abbreviations.

Line 221-Complete the sentence

 

 

Author Response

Reviewer #1: In this review, the authors present the latest research advances in the hydroxylation of natural products such as amino acids, steroids, terpenoids, lipids and phenylpropanoids. The manuscript contains a large number of references that provide a clear insight into the importance of this process as well as the application of the latest biotechnological approaches. However, the structure of the manuscript has numerous shortcomings.

Comment 1: Table 1 is completely uninformative, while the biosynthesis of hydroxylated amino acids is only presented in Table 2 in the rest of the text. Delete Table 1 and present the most important results for each group of natural products separately in tables.

Response: Thank you for your comments. We have made revisions based on your recommendation.We have added tables 2, 3, 4, 5.

In line 349 on page 9, in line 414 on page11, in line 461 on page 14, in line 511 on page 15, added Tables 2, 3, 4, and 5, respectively.

Table 2. Overview of the biosynthesis of hydroxylated steroids from different microbial strains.

Product	Chemical Structure	Host	Enzymes	Engineered Strategy	Titer	Reference
Testosterone		P. pastoris	17β-HSD3	Optimization of the gene codons of human 17β-HSD3	11.6 g/L	[133]
2α-Hydroxylated steroids		E. coli	CYP154C2	Rational engineering	15-20 mg/L	[33]
11α-OH-4AD		S. cerevisiae or Aspergillus oryzae	CYP68N1	Introducing a rapid identification strategy for filamentous fungi P450 enzymes	0.845 g/L	[34]
14α-OH-AD, 14α-OH-RSS		S. cerevisiae	P-450lun	Increasing the copies of P-450lun and CPRlun	150 mg/L, 64 mg/L	[118]

 

Table 3. Overview of the biosynthesis of hydroxylated terpenoids from different microbial strains.

Product	Chemical Structure	Host	Enzymes	Engineered Strategy	Titer	Reference
2-Hydroxy-dehydroabietic acid		S. cerevisiae	CYP72D19	Molecular docking and site-directed mutagenesis were used.	-	[155]
Trihydroxylated diterpene cyclooctatin		E. coli	P450s CotB3/CotB4	A new reductase/ferredoxin system was identified and characterized	15 mg/L	[145]
Ursolic acid, oleanolic acid, asiatic acid, madecassic acid, arjunolic acid		Y. lipolytica	CaCYP716C11p, CaCYP714E19p, and CaCYP716E41p.	Reconstructing the metabolic pathways in Y. lipolytica	11.6 mg/g, 10.2 mg/g, 0.12 mg/g, 8.9 mg/g, 4.4 mg/g	[156]
6β-Hydroxy-α-amyrin, 6β-hydroxy-β-amyrin, (2α,3β)-urs-12-ene-2,3-diol, (2α,3β)-olean-12-ene-2,3-diol, uvaol, erythrodiol		S. cerevisiae	EcOSC, CYP716E60, CYP716C80, CYP716A86 and CYP716A862.	Identified a oxidosqualene cyclase (EcOSC) gene and four CYP716 genes.	-	[149]

 

Table 4. Overview of the biosynthesis of hydroxylated lipids from different microbial strains.

Product	Chemical Structure	Host	Enzymes	Engineered Strategy	Titer	Reference
9,10-Dihydroxyhexadecanoic		E. coli	FAD, EH, EPOX	The heterologous expression of fatty acid hydroxylases.	-	[164]
ω-HFAs		S. cerevisiae	CYP52M1	Constructing a self-sufficient cytochrome P450 enzyme system	347 mg/L	[167]
ω-HPA		E. coli	CYP153A, camA/camB	By enhancing fatty acid synthesis, blocking the β-oxidation pathway, and optimizing NADH supply	610 mg/L	[175]

 

Table 5. Overview of the biosynthesis of hydroxylated phenylpropanoids from different microbial strains.

Product	Chemical Structure	Host	Enzymes	Engineered Strategy	Titer	Reference
Esculetin, piceatannol		E. coli	HpaBC	overexpression of HpaBC	2.7 g/L, 1.2 g/L.	[177]
Caffeic acid		S. cerevisiae	4HPA3H	A functional 4HPA3H was constructed to replace plant-derived cytochrome P450 enzymes.	289.4 mg/L	[190]
Eriodictyol, catechin, caffeic acid		E. coli	HpaBC	Optimization of media, induction temperature, induction point, and substrate delay time	62.7 mg/L, 34.7 mg/L, 3.5 g/L	[188]

 

Comment 2: The manuscript also lacks a section giving a brief overview of the bioengineering methods (metabolic engineering, enzyme engineering, genetic engineering, synthetic biology, artificial intelligence technologies) referred to by the authors.

Response: Thank you for your comments. We have made some revisions in the introduction.

In lines 93-117 on page 2：We have added one paragraph: " Biotechnological approaches are essential for the efficient biomanufacturing of hydroxylated natural products. Metabolic engineering alters the metabolic pathways of host cells and optimizes carbon flux to enhance the yield and productivity of target compounds. For instance, Song et al. [44] applied metabolic engineering to modify Escherichia coli, optimizing the biosynthetic pathways of 3-hydroxypropionic acid and malonic acid, thereby significantly increasing the fermentation yields of these hydroxylated compounds. Enzyme engineering utilizes techniques such as directed evolution and rational design to enhance the catalytic efficiency and specificity of hydroxylases. Cheng et al. [45] improved the catalytic performance of decarboxylase Chitinophage pinensis towards 3-hydroxylysine through semi-rational design and directed evolution, thereby enhancing the biosynthesis of the chiral amino alcohol 2-hydroxycadaverine. Genetic engineering techniques such as heterologous expression and promoter engineering enable precise control over key enzymes and regulatory elements. Lu et al. [46] enhanced the expression and activity of nicotinate dehydrogenase in non-model bacteria through heterologous expression and optimization of the expression system, achieving efficient biosynthesis of 6-hydroxy-3-cyanopyridine. Synthetic biology employs a modular and standardized approach to redesign biological systems, constructing efficient pathways for hydroxylation biosynthesis. Zhang et al. [47] employed synthetic biology to modularly assemble key metabolic modules including tryptophan hydroxylation, tryptophan biosynthesis, and cofactor regeneration, successfully constructing an artificial biosynthetic pathway from glucose to L-5-hydroxytryptophan (5-HTP). Additionally, emerging artificial intelligence technologies such as machine learning are being used to guide enzyme and metabolic engineering, accelerating the construction of hydroxylation cell factories [48,49]. The integrated application of biotechnological methods represents a vital pathway to achieving efficient biomanufacturing of hydroxylated natural products.”

Comment 3: A few illustrations showing the chemical formula of the compound and possible hydroxylation sites would contribute to the quality of the manuscript, especially for complex compounds such as steroids and their derivatives.

Response: Thank you for your excellent suggestions and comments. We have added the structural formulas of these compounds in the corresponding table based on your suggestion.

Comment 4: Keywords P450 monooxygenase-delete.

Response: Thank you for your comments. We have deleted this keyword-P450 monooxygenase in the revised manuscript.

Comment 5: Line 46 disease-malaria

Response: Thank you for your comments. We have made revisions based on your recommendation.

In line 43-45 on page 1, "For example, artemisinin, a natural product derived from the Artemisia annua plant, has become a standard drug for the treatment of this disease due to its excellent an-ti-malarial effects [11].” was revised as “For example, artemisinin, a natural product derived from the Artemisia annua plant, has become a standard drug for the treatment of this malaria due to its excellent an-ti-malarial effects [11].”

Comment 6: Line 113-The full name in front of CYP. Add one general sentence about these enzymes.

Response: Thank you for your comments. Sorry, we made such a mistake. We have made revisions based on your recommendation.

In lines 135-138 on page 4：We have added one general sentence: " Cytochrome P450 monooxygenases (CYPs) are a superfamily of versatile enzymes that catalyze a wide range of oxidative reactions, including the hydroxylation of various natural products and xenobiotics. They play crucial roles in the biosynthesis of sec-ondary metabolites and the metabolism of drugs and other foreign compounds [51].”

Comment 7: Line 130-Rewrite the beginning of the sentence.

Response: Thank you for your comments. We have made revisions based on your recommendation.

In line 156-159 on page 5, "This class of hydroxylases, featuring non-heme iron (II) as a cofactor and α-ketoglutarate as a co-substrate, can catalyze hydroxylation, cyclization, and desaturation reactions of substrates [60].” was revised as “Non-heme iron-dependent hydroxylases are another important class of enzymes involved in the hydroxylation of natural products. These hydroxylases feature non-heme iron (II) as a cofactor and α-ketoglutarate as a co-substrate, enabling them to catalyze hydroxylation, cyclization, and desaturation reactions of various substrates [60].”

Comment 8: Line 120-Escherichia coli (E. coli)-It is not necessary to put the abbreviations of the names of microorganism species in brackets. Review the entire manuscript. When microorganisms are mentioned, write the full name in the text first and then use the abbreviation.

Response: Thank you for your comments. Sorry, we made such a mistake. We have made revisions based on your recommendation.

Comment 9: Line 155-The full name in front of FMOs.

Response: Thank you for your comments. Sorry, we made such a mistake. We have made revisions based on your recommendation.

In line 183-184 on page 5, "such as FMOs and copper-dependent monooxygenases” was revised as “such as flavin-dependent monooxygenase (FMOs) and copper-dependent monooxygenases”.

Comment 10: Throughout the text, check that the full names are written before the abbreviations.

Response: Thank you for your comments. We have made revisions based on your recommendation.

Comment 11: Line 221-Complete the sentence.

Response: Thank you for your comments. We have made revisions based on your recommendation.

Sun et al. [6] established an in vitro multi-enzyme cascade catalytic system (MECCS) and a whole-cell catalytic system (WCCS) for the simultaneous production of succinic acid (SA) and 5-hydroxyleucine (5-HLeu), with a SA titer of 15.12 g/L and a 5-HLeu titer of 18.83 g/L.

Reviewer 2 Report

Comments and Suggestions for Authors

Comments:

The manuscript by Liu and coworkers summarized the recent progress of producing hydroxylating natural products. The authors focused on the hydroxylation of different classes of natural products including amino acids, terpenoids, fatty acid, steroids and phenylpropanoids. Overall, this review is well-written and I would recommend publication with minor changes to improve the clarity of the manuscript.

1.      Line 176. Based on the following examples, it appears that hydroxylated amino acids are often biosynthetic precursors of bioactive natural products and themselves do not usually exhibit antimicrobial activity. The authors may want to modify this sentence to make it more accurate.

2.      Line 242. The authors may want to specify the working volume of shake flask instead of using bottle.

3.      Line 381. The authors may want to specify the scale of fermentation to produce 260 mg and 290 mg of natural products.

4.      Line 439. The description of the structure is very confusing. What does “C6-C3 carbon skeleton” mean?

5.      The authors may consider including the chemical structures of some of the key hydroxylated natural products discussed in the manuscript. Providing these structures would help readers better visualize the compounds, as it can be difficult to fully grasp their structure based on names alone.

Author Response

Reviewer #2: The manuscript by Liu and coworkers summarized the recent progress of producing hydroxylating natural products. The authors focused on the hydroxylation of different classes of natural products including amino acids, terpenoids, fatty acid, steroids and phenylpropanoids. Overall, this review is well-written and I would recommend publication with minor changes to improve the clarity of the manuscript.

Comment 1: Line 176. Based on the following examples, it appears that hydroxylated amino acids are often biosynthetic precursors of bioactive natural products and themselves do not usually exhibit antimicrobial activity. The authors may want to modify this sentence to make it more accurate.

Response: Thank you for your comments. We have made revisions based on your recommendation.

In line 204-205 on page 5, "Hydroxylated amino acids (HAAs) are valued for their antiviral, antifungal, antibacterial, and anticancer properties” was revised as “Hydroxylated amino acids (HAAs) are important biosynthetic precursors of biologically active NPs, and are highly valued for their antiviral and anticancer properties”.

Comment 2: Line 242. The authors may want to specify the working volume of shake flask instead of using bottle.

Response: Thank you for your comments. Sorry, we made such a mistake. We have made revisions based on your recommendation.

In line 265-268 on page 7, "Klein et al. [99] established an in vivo biosynthetic pathway in E. coli for the preparation of various stereoisomers of hydroxyproline, including c3Hyp, c4Hyp, and t4Hyp, achieving yields of hydroxyproline up to 35-61% (175-305 mg/bottle) under shake flask culture conditions.” was revised as “Klein et al. [99] established an in vivo biosynthetic pathway in E. coli for the preparation of various stereoisomers of hydroxyproline, including c3Hyp, c4Hyp, and t4Hyp, achieving yields of hydroxyproline up to 35-61% (175–305 mg per shake flask) under shake flask culture conditions.”

Comment 3: Line 381. The authors may want to specify the scale of fermentation to produce 260 mg and 290 mg of natural products.

Response: Thank you for your comments. Sorry, we made such a mistake. We have made revisions based on your recommendation.

In line 402-405 on page 11, "The results indicated that the fungus could regioselectively oxidize the isopropyl group of the acyclic terpenoid compounds, producing 260 mg of hydroxycitronellol and 290 mg of 5-hydroxytetrahydrolavandulol, respectively.” was revised as “The results indicated that the fungus could regioselectively oxidize the isopropyl group of the acyclic terpenoid compounds in a 2 L fermentation medium, producing 260 mg of hydroxycitronellol and 290 mg of 5-hydroxytetrahydrolavandulol, respectively.”

Comment 4: Line 439. The description of the structure is very confusing. What does “C6-C3 carbon skeleton” mean?

Response: Thank you for your comments. The expression "C6-C3 carbon skeleton" in the original text may indeed lead to confusion among readers. We have made revisions based on your recommendation.

In line 463-467 on page 14, " Phenylpropanoids are a class of NPs with a C6-C3 carbon skeleton as the core structure, including flavonoids, stilbenes, and coumarins, which generally possess a phenolic structure” was revised as “Phenylpropanoids are a class of plant specialized metabolites synthesized from the amino acid phenylalanine through a series of enzymatic reactions. The core structure of phenylpropanoids consists of an aromatic ring (designated as C6) and a three-carbon side chain (designated as C3), which is typically a propionic acid or propanoid moiety. Many phenylpropanoids also possess phenolic hydroxyl groups attached to the aromatic ring”

Comment 5: The authors may consider including the chemical structures of some of the key hydroxylated natural products discussed in the manuscript. Providing these structures would help readers better visualize the compounds, as it can be difficult to fully grasp their structure based on names alone.

Response: Thank you for your comments. We have made revisions based on your recommendation.

Table 1. Overview of the biosynthesis of HAAs from different microbial strains.

Product	Chemical Structure	Host	Enzymes	Engineered Strategy	Titer	Reference
L-DOPA		E. coli	Fn-TPL	Overexpressin of a novel TPL	110 g/L	[101]
L-DOPA		C. glutamicum	Ralstonia solanacearum tyrosinase	Heterologous expression of Ralstonia solanacearum tyrosinase in C. glutamicum	0.61 g/g DCW	[102]
L-DOPA		E. coli	Eh-TPL	Enzyme evolution and high-throughput screening method	69.1 g/L	[103]
5-HTP		E. coli	TPH1	Heterologous expression of human tryptophan hydroxylase (TPH1) in E. coli	0.02 g/L	[104]
5-HTP		E. coli	TPH1	By protein engineering, manipulation of plasmid copy number, and fine-tuning of transcriptional regulation strategies	5.1 g/L	[105]
5-HTP		E. coli	TPH1	By rational design and molecular dynamics simulations	0.91 g/L	[106]
4-HIL		E. coli	IDO	By dynamically regulate ODHC activity	29.16 g/L	[107]
(2S,3R,4S)-4-HIL		E. coli	IDO	A high-throughput screening method was developed.	80.8 g/L/d	[108]
T-4-HYP		E. coli	P4H	Introduced a NOG pathway and redesigned key regulatory genes	89.4 g/L	[100]
T-4-HYP		E. coli	P4H	Heterologous expression of P4H from Dactylosporangium sp. strain RH1 in E. coli	41.0 g/L	[109]
4-HIL		C. glutamicum	IDO	By employing L-isoleucine-responsive transcriptional regulation or attenuation strategies	34.21 g/L	[93]

 

Table 2. Overview of the biosynthesis of hydroxylated steroids from different microbial strains.

Product	Chemical Structure	Host	Enzymes	Engineered Strategy	Titer	Reference
Testosterone		P. pastoris	17β-HSD3	Optimization of the gene codons of human 17β-HSD3	11.6 g/L	[133]
2α-hydroxylated steroids		E. coli	CYP154C2	Rational engineering	15-20 mg/L	[33]
11α-OH-4AD		S. cerevisiae or Aspergillus oryzae	CYP68N1	Introducing a rapid identification strategy for filamentous fungi P450 enzymes	0.845 g/L	[34]
14α-OH-AD, 14α-OH-RSS		S. cerevisiae	P-450lun	Increasing the copies of P-450lun and CPRlun	150 mg/L, 64 mg/L	[118]

 

Table 3. Overview of the biosynthesis of hydroxylated terpenoids from different microbial strains.

Product	Chemical Structure	Host	Enzymes	Engineered Strategy	Titer	Reference
2-hydroxy-dehydroabietic acid		S. cerevisiae	CYP72D19	Molecular docking and site-directed mutagenesis were used.	-	[155]
Trihydroxylated diterpene cyclooctatin		E. coli	P450s CotB3/CotB4	A new reductase/ferredoxin system was identified and characterized	15 mg/L	[145]
ursolic acid, oleanolic acid, asiatic acid, madecassic acid, arjunolic acid		Y. lipolytica	CaCYP716C11p, CaCYP714E19p, and CaCYP716E41p.	Reconstructing the metabolic pathways in Y. lipolytica	11.6 mg/g, 10.2 mg/g, 0.12 mg/g, 8.9 mg/g, 4.4 mg/g	[156]
6β-hydroxy-α-amyrin, 6β-hydroxy-β-amyrin, (2α,3β)-urs-12-ene-2,3-diol, (2α,3β)-olean-12-ene-2,3-diol, uvaol, erythrodiol		S. cerevisiae	EcOSC, CYP716E60, CYP716C80, CYP716A86 and CYP716A862.	Identified a oxidosqualene cyclase (EcOSC) gene and four CYP716 genes.	-	[149]

 

Table 4. Overview of the biosynthesis of hydroxylated lipids from different microbial strains.

Product	Chemical Structure	Host	Enzymes	Engineered Strategy	Titer	Reference
9,10-dihydroxyhexadecanoic		E. coli	FAD, EH, EPOX	The heterologous expression of fatty acid hydroxylases.	-	[164]
ω-HFAs		S. cerevisiae	CYP52M1	Constructing a self-sufficient cytochrome P450 enzyme system	347 mg/L	[167]
ω-HPA		E. coli	CYP153A, camA/camB	By enhancing fatty acid synthesis, blocking the β-oxidation pathway, and optimizing NADH supply	610 mg/L	[175]

 

Table 5. Overview of the biosynthesis of hydroxylated phenylpropanoids from different microbial strains.

Product	Chemical Structure	Host	Enzymes	Engineered Strategy	Titer	Reference
Esculetin, piceatannol		E. coli	HpaBC	overexpression of HpaBC	2.7 g/L, 1.2 g/L.	[177]
Caffeic acid		S. cerevisiae	4HPA3H	A functional 4HPA3H was constructed to replace plant-derived cytochrome P450 enzymes.	289.4 mg/L	[190]
Eriodictyol, catechin, caffeic acid		E. coli	HpaBC	Optimization of media, induction temperature, induction point, and substrate delay time	62.7 mg/L, 34.7 mg/L, 3.5 g/L	[188]

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Line 44-delete this in front of malaria