Designing Efficient Enzymes: Eight Predicted Mutations Convert a Hydroxynitrile Lyase into an Efficient Esterase.

Hydroxynitrile lyase from rubber tree (HbHNL) shares 45% identical amino acid residues with the homologous esterase from tobacco, SABP2, but the two enzymes catalyze different reactions. The x-ray structures reveal a serine-histidine-aspartate catalytic triad in both enzymes along with several differing amino acid residues within the active site. Previous exchange of three amino acid residues in the active site of HbHNL with the corresponding amino acid residue in SABP2 (T11G-E79H-K236M) created variant HNL3, which showed low esterase activity toward p-nitrophenyl acetate. Further structure comparison reveals additional differences surrounding the active site. HbHNL contains an improperly positioned oxyanion hole residue and differing solvation of the catalytic aspartate. We hypothesized that correcting these structural differences would impart good esterase activity on the corresponding HbHNL variant. To predict the amino acid substitutions needed to correct the structure, we calculated shortest path maps for both HbHNL and SABP2, which reveal correlated movements of amino acids in the two enzymes. Replacing four amino acid residues (C81L-N104T-V106F-G176S) whose movements are connected to the movements of the catalytic residues yielded variant HNL7TV (stabilizing substitution H103V was also added), which showed an esterase catalytic efficiency comparable to that of SABP2. The x-ray structure of an intermediate variant, HNL6V, showed an altered solvation of the catalytic aspartate and a partially corrected oxyanion hole. This dramatic increase in catalytic efficiency demonstrates the ability of shortest path maps to predict which residues outside the active site contribute to catalytic activity.

Identifying Key Residues in Lysine Decarboxylase for Soluble Expression Using Consensus Design Soluble Mutant Screening (ConsenSing).

Although recent advances in deep learning approaches for protein engineering have enabled quick prediction of hot spot residues improving protein solubility, the predictions do not always correspond to an actual increase in solubility under experimental conditions. Therefore, developing methods that rapidly confirm the linkage between computational predictions and empirical results is essential to the success of improving protein solubility of target proteins. Here, we present a simple hybrid approach to computationally predict hot spots possibly improving protein solubility by sequence-based analysis and empirically explore valuable mutants using split GFP as a reporter system. Our approach, Consensus design Soluble Mutant Screening (ConsenSing), utilizes consensus sequence prediction to find hot spots for improvement of protein solubility and constructs a mutant library using Darwin assembly to cover all possible mutations in one pot but still keeps the library as compact as possible. This approach allowed us to identify multiple mutants of Escherichia coli lysine decarboxylase, LdcC, with substantial increases in soluble expression. Further investigation led us to pinpoint a single critical residue for the soluble expression of LdcC and unveiled its mechanism for such improvement. Our approach demonstrated that following a protein's natural evolutionary path provides insights to improve protein solubility and/or increase protein expression by a single residue mutation, which can significantly change the profile of protein solubility.

Enhanced thermal stability of the ß-galactosidase BgaB from Bacillus circulans by cyclization mediated via SpyTag/SpyCatcher interaction and its use in galacto-oligosaccharides synthesis.

Cyclization of proteins using SpyTag/SpyCatcher is a novel approach to increase their thermal stability. In this paper, we test this approach on two ß-galactosidases from Bacillus circulans, BgaB and BgaC, and find that BgaB was stabilized while BgaC was not. Wild-type BgaB precipitated completely upon heating above 70 °C, but after SpyRing cyclization, it remained soluble after heating to 90 °C. Similarly, wild-type BgaB retained only 50 % activity after heating at 60 °C for 10 min, but this increased to 80 % after SpyRing cyclization. In contrast, cyclization decreased the stability of BgaC. After SpyRing cyclization, BgaC only retained 2 % activity after 20-min incubation at 55 °C, whereas the wild-type BgaC retained 25 % activity. One reason for the different effect of cyclization may the shorter distance between the N- and C-termini in BgaB (20.2 Å) as compared to BgaC (43.7 Å). The intrinsic fluorescence and circular dichroism spectra suggested that SpyRing cyclization of BgaB did not significantly change its conformation or secondary structure. SpyRing cyclized BgaB yielded similar amounts and compositions of galacto-oligosaccharides using a high initial lactose concentration (40 %, w/v), but a slightly higher amount at low initial lactose concentration (5 %, w/v) suggesting increased transgalactosylation activity.

Enzymatic Enantioselective anti-Markovnikov Hydration of Aryl Alkenes.

The addition of water to alkenes is an important method for the synthesis of alcohols, but the regioselectivity of acid-catalyzed hydration of terminal alkenes yields secondary alcohols according to Markovnikov's rule, making it difficult to obtain primary alcohols. Here we report a styrene monooxygenase that catalyzes the anti-Markovnikov hydration of the terminal aryl alkenes under anaerobic conditions. This hydration provides primary alcohols in good yields (up to 100 %), excellent anti-Markovnikov regioselectivity (>99 : 1), and good enantiomeric purity (60-83 % ee). Residues Asn46, Asp100, and Asn309 are essential for catalysis suggesting an acid-base mechanism with a carbanion-like intermediate that could account for the anti-Markovnikov regioselectivity. Our work reveals a new enzymatic tool with unusual regioselectivity based on the promiscuous catalytic activity of a monooxygenase.

Plasmid hypermutation using a targeted artificial DNA replisome.

Extensive exploration of a protein's sequence space for improved or new molecular functions requires in vivo evolution with large populations. But disentangling the evolution of a target protein from the rest of the proteome is challenging. Here, we designed a protein complex of a targeted artificial DNA replisome (TADR) that operates in live cells to processively replicate one strand of a plasmid with errors. It enhanced mutation rates of the target plasmid up to 2.3 × 105-fold with only a 78-fold increase in off-target mutagenesis. It was used to evolve itself to increase error rate and increase the efficiency of an efflux pump while simultaneously expanding the substrate repertoire. TADR enables multiple simultaneous substitutions to discover functions inaccessible by accumulating single substitutions, affording potential for solving hard problems in molecular evolution and developing biologic drugs and industrial catalysts.

High-Level Production of Lysine in the Yeast Saccharomyces cerevisiae by Rational Design of Homocitrate Synthase.

Homocitrate synthase (HCS) catalyzes the aldol condensation of 2-oxoglutarate (2-OG) and acetyl coenzyme A (AcCoA) to form homocitrate, which is the first enzyme of the lysine biosynthetic pathway in the yeast Saccharomyces cerevisiae. The HCS activity is tightly regulated via feedback inhibition by the end product lysine. Here, we designed a feedback inhibition-insensitive HCS of S. cerevisiae (ScLys20) for high-level production of lysine in yeast cells. In silico docking of the substrate 2-OG and the inhibitor lysine to ScLys20 predicted that the substitution of serine with glutamate at position 385 would be more suitable for desensitization of the lysine feedback inhibition than the substitution from serine to phenylalanine in the already known Ser385Phe variant. Enzymatic analysis revealed that the Ser385Glu variant is far more insensitive to feedback inhibition than the Ser385Phe variant. We also found that the lysine contents in yeast cells expressing the Ser385Glu variant were 4.62- and 1.47-fold higher than those of cells expressing the wild-type HCS and Ser385Phe variant, respectively, due to the extreme desensitization to feedback inhibition. In this study, we obtained highly feedback inhibition-insensitive HCS using in silico docking and enzymatic analysis. Our results indicate that the rational engineering of HCS for feedback inhibition desensitization by lysine could be useful for constructing new yeast strains with higher lysine productivity. IMPORTANCE A traditional method for screening toxic analogue-resistant mutants has been established for the breeding of microbes that produce high levels of amino acids, including lysine. However, another efficient strategy is required to further improve their productivity. Homocitrate synthase (HCS) catalyzes the first step of lysine biosynthesis in the yeast Saccharomyces cerevisiae, and its activity is subject to feedback inhibition by lysine. Here, in silico design of a key enzyme that regulates the biosynthesis of lysine was utilized to increase the productivity of lysine. We designed HCS for the high-level production of lysine in yeast cells by in silico docking simulation. The engineered HCS exhibited much less sensitivity to lysine and conferred higher production of lysine than the already known variant obtained by traditional breeding. The combination of in silico design and experimental analysis of a key enzyme will contribute to advances in metabolic engineering for the construction of industrial microorganisms.

Consensus Finder web tool to predict stabilizing substitutions in proteins.

The consensus sequence approach to predicting stabilizing substitutions in proteins rests on the notion that conserved amino acids are more likely to contribute to the stability of a protein fold than non-conserved amino acids. To implement a prediction for a target protein sequence, one finds homologous sequences and aligns them in a multiple sequence alignment. The sequence of the most frequently occurring amino acid at each position is the consensus sequence. Replacement of a rarely occurring amino acid in the target with a frequently occurring amino acid from the consensus sequence is predicted to be stabilizing. Consensus Finder is an open-source web tool that automates this prediction. This chapter reviews the rationale for the consensus sequence approach and explains the options for fine-tuning this approach using Staphylococcus nuclease A as an example.

Larger active site in an ancestral hydroxynitrile lyase increases catalytically promiscuous esterase activity.

Hydroxynitrile lyases (HNL's) belonging to the α/ß-hydrolase-fold superfamily evolved from esterases approximately 100 million years ago. Reconstruction of an ancestral hydroxynitrile lyase in the α/ß-hydrolase fold superfamily yielded a catalytically active hydroxynitrile lyase, HNL1. Several properties of HNL1 differ from the modern HNL from rubber tree (HbHNL). HNL1 favors larger substrates as compared to HbHNL, is two-fold more catalytically promiscuous for ester hydrolysis (p-nitrophenyl acetate) as compared to mandelonitrile cleavage, and resists irreversible heat inactivation to 35 °C higher than for HbHNL. We hypothesized that the x-ray crystal structure of HNL1 may reveal the molecular basis for the differences in these properties. The x-ray crystal structure solved to 1.96-Å resolution shows the expected α/ß-hydrolase fold, but a 60% larger active site as compared to HbHNL. This larger active site echoes its evolution from esterases since related esterase SABP2 from tobacco also has a 38% larger active site than HbHNL. The larger active site in HNL1 likely accounts for its ability to accept larger hydroxynitrile substrates. Site-directed mutagenesis of HbHNL to expand the active site increased its promiscuous esterase activity 50-fold, consistent with the larger active site in HNL1 being the primary cause of its promiscuous esterase activity. Urea-induced unfolding of HNL1 indicates that it unfolds less completely than HbHNL (m-value = 0.63 for HNL1 vs 0.93 kcal/mol·M for HbHNL), which may account for the ability of HNL1 to better resist irreversible inactivation upon heating. The structure of HNL1 shows changes in hydrogen bond networks that may stabilize regions of the folded structure.

Evolutionary innovation using EDGE, a system for localized elevated mutagenesis.

Mutations arising across the whole genome can hinder the emergence of evolutionary innovation required for adaptation because many mutations are deleterious. This trade-off is overcome by elevated mutagenesis to localized loci. Examples include phase variation and diversity-generating retroelements. However, these mechanisms are rare in nature; and all have narrow mutational spectra limiting evolutionary innovation. Here, we engineer a platform of Experimental Designed Genic Evolution (EDGE) to study the potential for evolutionary novelty at a single locus. Experimental evolution with EDGE shows that bacterial resistance to a novel antibiotic readily evolves, provided that elevated mutagenesis is focused on a relevant gene. A model is proposed to account for the cost and benefit of such single loci to adaptation in a changing environment and explains their high mutation rates, limited innovation, and the rarity of localized mutagenesis in nature. Overall, our results suggest that localized mutation systems can facilitate continuing adaptive evolution without necessarily restricting the spectrum of mutations. EDGE has utility in dissecting the complex process of adaptation with its localized, efficient evolution.

Engineering more stable proteins.

Protein function requires the folded protein form, but this form is unstable mainly because it readily unfolds into a flexible, unstructured form. Protein folding is favored by burying of hydrophobic side chains and hydrogen bonding between the amino acids. Protein unfolding is favored by the increase in conformational freedom of the main chain of amino acids upon unfolding. Protein stability is usually measured by the reversible unfolding of the protein with either heat or chemical additives like urea. Engineering mores stable proteins involves making substitutions that shift the folding-unfolding balance toward the folded form. Stabilizing substitutions can either stabilize the folded conformation or destabilize the unfolded ensemble. This tutorial emphasizes web-based tools to identify substitutions that stabilize proteins. Besides unfolding, other sources of protein instability are chemical modifications like oxidations or cleavage by proteases and aggregation of partly unfolded proteins into insoluble particles.

Developmental evolution facilitates rapid adaptation.

Developmental evolution has frequently been identified as a mode for rapid adaptation, but direct observations of the selective benefits and associated mechanisms of developmental evolution are necessarily challenging to obtain. Here we show rapid evolution of greatly increased rates of dispersal by developmental changes when populations experience stringent selection. Replicate populations of the filamentous fungus Trichoderma citrinoviride underwent 85 serial transfers, under conditions initially favoring growth but not dispersal. T. citrinoviride populations shifted away from multicellular growth toward increased dispersal by producing one thousand times more single-celled asexual conidial spores, three times sooner than the ancestral genotype. Conidia of selected lines also germinated fifty percent faster. Gene expression changed substantially between the ancestral and selected fungi, especially for spore production and growth, demonstrating rapid evolution of tight regulatory control for down-regulation of growth and up-regulation of conidia production between 18 and 24 hours of growth. These changes involved both developmentally fixed and plastic changes in gene expression, showing that complex developmental changes can serve as a mechanism for rapid adaptation.

Comparison of Five Protein Engineering Strategies for Stabilizing an α/ß-Hydrolase.

A review of the previous stabilization of α/ß-hydrolase fold enzymes revealed many different strategies, but no comparison of strategies on the same enzyme. For this reason, we compared five strategies to identify stabilizing mutations in a model α/ß-hydrolase fold enzyme, salicylic acid binding protein 2, to reversible denaturation by urea and to irreversible denaturation by heat. The five strategies included one location agnostic approach (random mutagenesis using error-prone polymerase chain reaction), two structure-based approaches [computational design (Rosetta, FoldX) and mutation of flexible regions], and two sequence-based approaches (addition of proline at locations where a more stable homologue has proline and mutation to consensus). All strategies identified stabilizing mutations, but the best balance of success rate, degree of stabilization, and ease of implementation was mutation to consensus. A web-based automated program that predicts substitutions needed to mutate to consensus is available at http://kazlab.umn.edu .

Biosynthesis of (-)-5-Hydroxy-equol and 5-Hydroxy-dehydroequol from Soy Isoflavone, Genistein Using Microbial Whole Cell Bioconversion.

Equols are isoflavandiols formed by reduction of soy isoflavones such as daidzein and genistein by gut microorganisms. These phytoestrogens are of interest for their various biological effects. We report biosynthesis from genistein to (-)-5-hydroxy-equol in recombinant E. coli expressing three reductases (daidzein reductase DZNR, dihidrodaidzein reductase DHDR, tetrahydrodaidzein reductase THDR) and a racemase (dihydrodaidzein racemase, DDRC) originating from the gut bacterium, Slackia isoflavoniconvertens. The biosynthesized 5-hydroxy-equol proved as an optically negative enantiomer, nonetheless it displayed an inverse circular dichroism spectrum to (S)-equol. Compartmentalized expression of DZNR and DDRC in one E. coli strain and DHDR and THDR in another increased the yield to 230 mg/L and the productivity to 38 mg/L/h. If the last reductase was missing, the intermediate spontaneously dehydrated to 5-hydroxy-dehydroequol in up to 99 mg/L yield. This novel isoflavene, previously not known to be synthesized in nature, was also detected in this biotransformation system. Although (S)-equol favors binding to human estrogen receptor (hER) ß over hERα, (-)-5-hydroxy-equol showed the opposite preference. This study provides elucidation of the biosynthetic route of (-)-5-hydroxy-equol and measurement of its potent antagonistic character as a phytoestrogen for the first time.

One-step pretreatment of yellow poplar biomass using peracetic acid to enhance enzymatic digestibility.

Pretreatment of biomass with dilute acid requires high temperatures of >160 °C to remove xylan and does not remove lignin. Here we report that the addition of peracetic acid, a strong oxidant, to mild dilute acid pretreatment reduces the temperature requirement to only 120 °C. Pretreatment of yellow poplar with peracetic acid (300 mM, 2.3 wt%) and dilute sulfuric acid (100 mM, 1.0 wt%) at 120 °C for 5 min removed 85.7% of the xylan and 90.4% of the lignin leaving a solid consisting of 75.6% glucan, 6.0% xylan and 4.7% lignin. Low enzyme loadings of 5 FPU/g glucan and 10 pNPGU/g glucan converted this solid to glucose with an 84.0% yield. This amount of glucose was 2.5 times higher than with dilute acid-pretreated solid and 13.8 times higher than with untreated yellow poplar. Thus, the addition of peracetic acid, easily generated from acetic acid and hydrogen peroxide, dramatically increases the effectiveness of dilute acid pretreatment of biomass.

Identical active sites in hydroxynitrile lyases show opposite enantioselectivity and reveal possible ancestral mechanism.

Evolutionarily related hydroxynitrile lyases from rubber tree (HbHNL) and from Arabidopsis thaliana (AtHNL) follow different catalytic mechanisms with opposite enantioselectivity toward mandelonitrile. We hypothesized that the HbHNL-like mechanism evolved from an enzyme with an AtHNL-like mechanism. We created ancestor-like composite active-sites in each scaffold to elucidate how this transition may have occurred. Surprisingly, a composite active site in HbHNL maintained (S)-selectivity, while the identical set of active site residues in AtHNL maintained (R)-selectivity. Composite active-site mutants that are (S)-selective without the Lys236 and Thr11 that are required for the classical (S)-HNL mechanism suggests a new mechanism. Modeling suggested a possibility for this new mechanism that does not exist in modern enzymes. Thus, the last common ancestor of HbHNL and AtHNL may have used an extinct mechanism, not the AtHNL-like mechanism. Multiple mechanisms are possible with the same catalytic residues and residues outside the active site strongly influence mechanism and enantioselectivity.

The Fungus Trichoderma Regulates Submerged Conidiation Using the Steroid Pregnenolone.

In previous work, we evolved a population of Trichoderma citrinoviride in liquid cultures to speed up its asexual development cycle. The evolved population, called T-6, formed conidia 3 times sooner and in >1000-fold greater numbers. Here, we identify the steroid pregnenolone as a molecular signal for this different behavior. Media in which the ancestral T. citrinoviride population was grown (called ancestral spent media) contained a submerged conidiation inhibitor. Growing the evolved population T-6 in ancestral spent media eliminated the abundant formation of conidia. This inhibition depended on the amount and age of the ancestral spent medium and the time that the ancestral spent medium was added to the T-6 culture. Fractionation of the ancestral spent medium identified a hydrophobic inhibiting compound with a molecular weight less than 2000 g/mol. A combination of GC-MS, ELISA, and reaction with cholesterol oxidase identified it as pregnenolone. The addition of pregnenolone to cultures of T-6 inhibited submerged conidiation by inhibiting formation of conidiophores, while 10 other analogous steroids did not. Pregnenolone also inhibited submerged conidiation of Fusarium graminearum PH-1, a plant pathogen that causes head blight in wheat and barley. This identification of steroids as signal molecules in fungi creates opportunities to disrupt this signaling to control fungal behavior.

Experimental Evolution of Trichoderma citrinoviride for Faster Deconstruction of Cellulose.

Engineering faster cellulose deconstruction is difficult because it is a complex, cooperative, multi-enzyme process. Here we use experimental evolution to select for populations of Trichoderma citrinoviride that deconstruct up to five-fold more cellulose. Ten replicate populations of T. citrinoviride were selected for growth on filter paper by serial culture. After 125 periods of growth and transfer to fresh media, the filter paper deconstruction increased an average of 2.5 fold. Two populations were examined in more detail. The activity of the secreted cellulase mixtures increased more than two-fold relative to the ancestor and the largest increase was in the extracellular ß-glucosidase activity. qPCR showed at least 16-fold more transcribed RNA for egl4 (endoglucanase IV gene), cbh1 (cellobiohydrolase I gene) and bgl1 (extracellular ß-glucosidase I gene) in selected populations as compared to the ancestor, and earlier peak expressions of these genes. Deep sequencing shows that the regulatory strategies used to alter cellulase secretion differ in the two strains. The improvements in cellulose deconstruction come from earlier expression of all cellulases and increased relative amount of ß-glucosidase, but with small increases in the total secreted protein and therefore little increase in metabolic cost.

Catalytic Promiscuity of Ancestral Esterases and Hydroxynitrile Lyases.

Catalytic promiscuity is a useful, but accidental, enzyme property, so finding catalytically promiscuous enzymes in nature is inefficient. Some ancestral enzymes were branch points in the evolution of new enzymes and are hypothesized to have been promiscuous. To test the hypothesis that ancestral enzymes were more promiscuous than their modern descendants, we reconstructed ancestral enzymes at four branch points in the divergence hydroxynitrile lyases (HNL's) from esterases ∼ 100 million years ago. Both enzyme types are α/ß-hydrolase-fold enzymes and have the same catalytic triad, but differ in reaction type and mechanism. Esterases catalyze hydrolysis via an acyl enzyme intermediate, while lyases catalyze an elimination without an intermediate. Screening ancestral enzymes and their modern descendants with six esterase substrates and six lyase substrates found higher catalytic promiscuity among the ancestral enzymes (P < 0.01). Ancestral esterases were more likely to catalyze a lyase reaction than modern esterases, and the ancestral HNL was more likely to catalyze ester hydrolysis than modern HNL's. One ancestral enzyme (HNL1) along the path from esterase to hydroxynitrile lyases was especially promiscuous and catalyzed both hydrolysis and lyase reactions with many substrates. A broader screen tested mechanistically related reactions that were not selected for by evolution: decarboxylation, Michael addition, γ-lactam hydrolysis and 1,5-diketone hydrolysis. The ancestral enzymes were more promiscuous than their modern descendants (P = 0.04). Thus, these reconstructed ancestral enzymes are catalytically promiscuous, but HNL1 is especially so.

Evolution of a Catalytic Mechanism.

The means by which superfamilies of specialized enzymes arise by gene duplication and functional divergence are poorly understood. The escape from adaptive conflict hypothesis, which posits multiple copies of a gene encoding a primitive inefficient and highly promiscuous generalist ancestor, receives support from experiments showing that resurrected ancestral enzymes are indeed more substrate-promiscuous than their modern descendants. Here, we provide evidence in support of an alternative model, the innovation-amplification-divergence hypothesis, which posits a single-copied ancestor as efficient and specific as any modern enzyme. We argue that the catalytic mechanisms of plant esterases and descendent acetone cyanohydrin lyases are incompatible with each other (e.g., the reactive substrate carbonyl must bind in opposite orientations in the active site). We then show that resurrected ancestral plant esterases are as catalytically specific as modern esterases, that the ancestor of modern acetone cyanohydrin lyases was itself only very weakly promiscuous, and that improvements in lyase activity came at the expense of esterase activity. These observations support the innovation-amplification-divergence hypothesis, in which an ancestor gains a weak promiscuous activity that is improved by selection at the expense of the ancestral activity, and not the escape from adaptive conflict in which an inefficient generalist ancestral enzyme steadily loses promiscuity throughout the transition to a highly active specialized modern enzyme.

Production of p-hydroxybenzoic acid from p-coumaric acid by Burkholderia glumae BGR1.

p-Coumaric acid (pCA) is abundant in biomass with low lignin content, such as straw and stubble from rye, wheat, and barley. pCA can be isolated from biomass and used for the synthesis of aromatic hydrocarbons. Here, we report engineering of the natural pathway for conversion of pCA into p-hydroxybenzoic acid (pHBA) to increase the amount of pHBA that accumulates more than 100-fold. Burkholderia glumae strain BGR1 (BGR1) grows efficiently on pCA as a sole carbon source via a CoA-dependent non-ß-oxidation pathway. This pathway removes two carbons from pCA as acetyl-CoA yielding p-hydroxybenzaldehyde and subsequently oxidizes it to pHBA. To increase the amount of accumulated pHBA in BGR1, we first deleted two genes encoding enzymes that degrade pHBA in the ß-ketoadipate pathway. At 10 mM of pCA, the double deletion mutant BGR1_PB4 (Δphb3hΔbcl) accumulated pHBA with 95% conversion, while the control BGR1 accumulated only with 11.2% conversion. When a packed bed reactor containing immobilized BGR1_PB4 cells was operated at a dilution rate 0.2 h(-1) , the productivity of pHBA was achieved at 9.27 mg/L/h for 134 h. However, in a batch reactor at 20 mM pCA, growth of BGR1_PB4 was strongly inhibited, resulting in a low conversion of 19.3%. To further increase the amount of accumulated pCA, we identified the first enzyme in the pathway, p-hydroxcinnmaoyl-CoA synthetase II (phcs II), as the rate-limiting enzyme. Over expression of phcs II using a Palk promoter in a batch reaction at 20 mM of pCA yielded 99.0% conversion to pHBA, which is the highest concentration of pHBA ever reported using a biological process. Biotechnol. Bioeng. 2016;113: 1493-1503. © 2015 Wiley Periodicals, Inc.