<p>Online Resource 1</p><p>1 A process model for rifamycin B has been described in detail recently (Bapat et al.,</p><p>2006a). The model is based on cybernetic principles and assumes that in complex</p><p> medium the organism has access to up to three substrate combination: (i)</p><p>Combination I: uptake of AA [Equation (A.1a), (A.1b) for stoichiometry and</p><p> growth rate on this substrate], (ii) Combination II: simultaneous uptake of AA (S1)</p><p> and glucose (S2) [ Equation (A.2a), (A.2b)] and (iii) Combination III:</p><p> simultaneous uptake of glucose (S2) and AMS (S3) [Equation (A.3a), (A.3b)]. It is</p><p> assumed that the free AA is released by hydrolysis of proteins and peptides (S 4)</p><p>[Equation (A.4a), (A.4b)]. Equation (A.1a), (A.1b) – (A.4a), (A.4b) represents the</p><p> four main growth reactions with each equation consisting of the stoichiometry</p><p> followed by the kinetic model form. The uptake of substrate combination ‘i’ is</p><p> dependent on the level of the corresponding key enzyme (XEi), which may be</p><p> inducible. Thus, the organism invests resources to synthesize XEi based on the</p><p> growth achievable on the corresponding substrate combination. Although a unique</p><p> growth rate exists corresponding to each substrate combination, the overall</p><p> specific growth rate, μ, and specific production rate, qp, are the weighted sums of</p><p> the specific growth and production rates on the individual substrate combination.</p><p>The weights αi are estimated using the optimality criteria [Equation (A.8)] in</p><p> which the organism allows the uptake of all the substrate combinations as long as</p><p> the specific growth rate obtained by summing the rates on different substrate</p><p> combination is less than its intrinsic growth capacity denoted by μmax. The</p><p> complete model comprises of component mass balances represented by ordinary</p><p> differential equation (ODE’s) [Equation (A.9) – (A.16)] along with the optimality</p><p>2 criteria stated in Equation (A.8). The simulation exercise to predict growth,</p><p> product formation and substrate utilization was carried out by integrating the set</p><p> of simultaneous differential equations as an initial value problem. The model</p><p> parameters and bounds or nominal values of the process variables are listed in</p><p>Table I and II, respectively. </p><p>The key features of the model include:</p><p>. The nitrogen catabolite repression of rifamycin B formations [Equation (A.21),</p><p>(A.22)].</p><p>. The sequential utilization of substrates in a complex multi-substrate media</p><p>[Equation (A.8)].</p><p>. The model captures the fact that rifamycin B formation does not occur when AA</p><p> are taken up as sole source of carbon and nitrogen.</p><p>. The model considers inhibition of AA utilizing enzyme (XE1) by glucose (S2)</p><p> due to carbon catabolite repression [Equation (A.15)].</p><p>. The enzymes (XEi) are inducible and are assumed to undergo degradation via first</p><p> order kinetics [Equation (A.15) and (A.16)]. </p><p>For greater details about the model, the interested reader may refer to (Bapat et al.,</p><p>2006a). The model equations are summarized below.</p><p>Amino acid uptake:</p><p>Equation “a” is used to represent stoichiometry and Equation b is used to represent the kinetic form.</p><p>3 X  Y1,3CO 2 - Y1,1 S1  Y1,4O2  Y1,5 H 2O 0 (A.1a)</p><p> max  X  * μ  α μ  E1  r (A.2b) 1 1 1  X  1  E1 ,Re f </p><p>Simultaneous uptake of AA and glucose:</p><p>X  Y2,3CO 2 - Y2,1 S1  Y2,2 S2  Y2,4O2  Y2,5H 2O 0 (A.2a)</p><p> max  X  * μ  α μ  E 2  r (A.2b) 2 2 2  X  2  E 2 ,Re f </p><p>Simultaneous uptake of AMS and glucose: </p><p>X  Y3,3CO 2 - Y3,3 S3  Y3,2 S2  Y3,4O2  Y3,5H 2O 0 (A.3a)</p><p> max  X  * μ  α μ  E3  r (A.3b) 3 3 3  X  3  E3 ,Re f </p><p>Conversion of insoluble nitrogen to AA:</p><p>S1  S4 0 (A.4a)</p><p> X E  r   4  r* k X (A.4b) diss.  X  4 4  E4 , Re f </p><p>Rifamycin B production:</p><p>P  Y5,3CO 2 - Y5,1 S1  Y5,2 S2  Y5,4O2  Y5,5 H 2O 0 (A.5a)</p><p> max  X  qp  α qp  E 2  r* (A.5b) 2 2 2  X  2,P  E 2 ,Re f </p><p>P  Y6,3CO 2 - Y6,3 S3  Y6,2 S2  Y6,4O 2  Y6,5H 2O 0 (A.6a)</p><p>4 max  X  qp  α qp  E 3  r* (A.6b) 3 3 3  X  3,P  E3 ,Re f </p><p>Enzyme synthesis:</p><p>X  X 0 Ei (A.7a) r  K r* Ei Ei i ; For i = 1, 2, 3 (A.7b)</p><p>Optimality criteria:</p><p> max(μ),</p><p>,0  αi  1, (A.8) s.t. and μ  μmax</p><p>3 Where μ  αiμi i1</p><p>Mass Balance Equations</p><p>Biomass: dX   .X ; (A.9) dt</p><p>Amino acids: d( S ) 骣 S 1 =F C -轾 ym a + y m + y q a X + k E 琪 4 (A.10) S1 FS 2 臌( 1,1 1) 1( 2,1 2 5,1 P ,2) 2 4 ( prot ) dt桫 kS 4+ S 4</p><p>Glucose: dS  2  F C  y   y q   y   y q  X (A.11) dt S 2 FS 2 2,2 2 5,2 P,2 2 3,2 3 6,2 P,3 3</p><p>Ammonium Sulfate: dS  3  F C  y   y q  X (A.12) dt S 3 FS 3 3,3 3 6,3 P,3 3</p><p>5 Insoluble nitrogen: dS   S  4  F C  y   y   y q  X  k E  4  (A.13) S 4 FS 4 1,1 1 2,1 2 5,1 P,2 4 prot   dt  kS 4  S4  dP 3  qP .X ; Where q P  αi q P i (A.14) dt i1</p><p>Enzymes:</p><p>X E d 1 X X E E 1,Ref max S1   1 (A.15) = (μ  k )  μ  k  dt i deg,i S 2  deg,i  X S  Ks  S  1 E 2 1,1 1 1,Ref KI1,1</p><p>X E d i X X E E i,Ref max *   i (i = 2, 3) (A.16) = (μ  k ) r  μ  k  dt i deg,i i  deg,i  X E i, Ref</p><p>* Where, ri indicate the effect of substrate limitation on growth and product formation and are given by,</p><p>Growth on amino acids,</p><p>* S1 r1  2 S1 (A.17) Ks1,1  S1  K I1,1</p><p>Growth on substrate combination, 2 (Fig. 3)</p><p>* S1 S2 r2  2 2 S1 S2 (A.18) Ks 2,1  S1  Ks 2,2  S2  K I 2,1 K I 2,2</p><p>Production on substrate combination, 3 (Fig. 3)</p><p>6 * S3 S2 r3  2 2 S3 S2 (A.19) Ks3,3  S3  Ks3,2  S2  K I3,3 K I 3,2</p><p>* S4 r4  (A.20) ks 4,1  S4</p><p>Production on substrate combination, 2 </p><p>* S1 S2 r2,P  2 2 S1 S2 (A.21) Kp5,1  S1  Kp  S  K 5,2 2 PI 5,1 K PI 5,2</p><p>Production on substrate combination, 3 </p><p>* S3 S2 r3,P  2 2 S3 S2 (A.22) Kp6,1  S3  Kp  S  K 6,2 2 PI 6,1 K PI 6,2</p><p>Some of the terms and parameters used in the equations have been explained below for clarity:- </p><p>αk : Control parameter that determines the metabolic flux through </p><p> branch k CFsi : Concentration of substrate combination i in the respective feed </p><p> stream Fsi Fsi : Feed flow rate of substrate combination i -1 Kdeg,i : Degradation constant for the enzyme Ei, h</p><p>Ksi, j and Kpi, j : Substrate half saturation constant for substrate j in the model for </p><p> reaction i, g L-1 -1 qp2 : Specific product formation rate on amino acid and glucose, h -1 qp3 : Specific product formation rate on AMS and glucose, h ri : Reaction rate for substrate combination i</p><p>7 max -1 1 : Specific growth rate on S1, h max -1 2 : Specific growth rate on S2 and S1, h max -1 3 : Specific growth rate on S2 and S3, h XEi : Concentration of the enzyme responsible for uptake of substrate </p><p> combination i. -1 Y i, j : Stoichiometric coefficient of substrate j in reaction i, g g .</p><p>8</p>